New insights into the regulation of TGF‐β/Smad and MPK signaling pathway gene expressions by nasal allergen and methacholine challenge test in asthma

Abstract Background Asthma is a heterogeneous chronic inflammatory disease of the bronchi, the course of which is significantly influenced by extrinsic factors (specific and non‐specific). Methods The aim of this study was to evaluate the effect of these factors represented by nasal allergen challenge (specific factors) and methacholine challenge test (non‐specific) on changes in mRNA expression of genes encoding the TGF‐β (TGF‐β1 and TGF‐β3)‒Smad (mitogen‐activated protein kinase 1/3 [MPK1/3], Smad1/3/6/7) signaling pathway in asthmatic patients. Results Seventy‐five subjects were included in the study, of whom 27 were applied an intranasal allergen provocation and 48 a methacholine provocation. There were 9 men and 18 women in the intranasal provocation group, and 17 men and 31 women in the methacholine test group. We found that both examined the types of challenges contributed to changes in the relative expression of genes of the TGF‐β (TGF‐β1 and TGF‐β3)‒Smad (MPK1/3, Smad1/3/6/7) signaling pathway in asthmatic patients. A decrease was noted for MAPK1, MAPK3, Smad3, Smad6, and Smad7 genes and an increase of up to 2.5 times for TGF‐β1 gene. Conclusions Our experiment allows us to conclude that the change in the mRNA expression of the TGF‐β1–MPK1/3 and Smad3/6/7 genes occurs after an intranasal allergen and bronchial methacholine challenge.


| INTRODUCTION
Asthma is a heterogeneous chronic inflammatory disease of the bronchi, the course of which is significantly influenced by extrinsic factors (specific and non-specific). [1][2][3] It should be pointed out that irrespective of the type of inducer of inflammation in asthma (specific factors, e.g.: allergens; non-specific factors, e.g.: pollutants, or other analogous/related substances) bronchial remodeling occurs as a result of TGF-β (Transforming Growth Factor beta) overexpression. [4][5][6] It is a profibrotic cytokine that is found in humans in three isoforms (TGFβ-1, TGFβ-2, TGFβ-3). It stimulates the process of growth and differentiation of many cell types, controls their proliferation and apoptosis, and stimulates fibroblasts and bronchial smooth muscle cells to control the metabolism of extracellular matrix (ECM) proteins. Production of TGF-β is associated with the presence of eosinophils in the airways of asthmatic patients. Eosinophilic granulocytes secrete some other profibrogenic molecules, such as eosinophil cationic protein (ECP). The TGF-β superfamily are considered to be a group of key mediators, playing a role in the regulation of allergic and non-allergic inflammation. It has a significant impact on airway remodeling in asthma. [7][8][9] Intracellular effectors of TGF-β signaling include among others: Smad proteins (Mothers Against Decapentaplegic, MAD and SMA gene; the name is a combination of the names of two homologous proteins Sma and MAD found in Caenorhabditis elegans and Drosophila melanogaster). They are activated by TGF-β receptors and travel to the cell nucleus, where they regulate the transcription of over 500 genes, including those responsible for bronchial remodeling. [10][11][12][13][14][15][16][17] TGF-β cytokine activates the TGFβRI/TGFβRII receptor Acti- TGFβRI/TGFβRII (ALK1) receptors is different and depends on the activation inducing factor: specific factors and non-specific factors.
Hence, the cellular response of TGF-β/Smad and mitogen-activated protein kinase (MPK) signal pathway proteins to the nasal allergen challenge and methacholine challenge test is especially important in the pathogenesis of asthma, including bronchial remodeling. 5,10,[14][15][16][17][18][19] In vitro and in vivo studies in asthma indicate that specific and non-specific provoking agents can induce bronchial remodeling independent of inflammation. Active provocations with allergen (which causes bronchospasm and eosinophilic inflammation) or methacholine (which causes bronchospasm without eosinophilic inflammation) are performed to show the effect of specific and non-specific factors on molecular underpinnings in patients with asthma. 5,10,14-19

| AIM
The aim of this study was to evaluate the effect of nasal allergen challenge and methacholine challenge test on changes in mRNA expression of genes encoding the TGF-β (TGF-β1 and TGF-β3)-Smad (MPK1/3, Smad1/3/6/7) signaling pathway in peripheral blood mononuclear cell (PBMC) of asthmatic patients.

| Study group
A convenience sample of in-and outpatients with asthma (diag- the survey questionnaire (medical questionnaire) were collected by specialists in internal medicine, allergology, and lung diseases. The patients who had been qualified to the study by specialists (given above), underwent intranasal allergen provocations or methacholine tests according to medical recommendations (diagnosis of allergic rhinitis or diagnosis of asthma) and in compliance with the current standards for such tests. Next, 9 ml of blood was collected from patients before the provocation (at the time coded 0 h) and two times after the provocation, at 1 and 24 h. Peripheral venous blood was collected from the ulnar vein. There were two separate patient cohorts in the study. One-patients had a nasal allergen challenge, and the other cohort-methacholine challenge.

| Asthma diagnosis
Asthma diagnosis was established according to The Global Initiative For Asthma (GINA) 2019 recommendations, based on clinical asthma symptoms and a lung function test. The level of asthma severity and control was determined on the basis of GINA Report Guidelines. All the participants underwent subjective examinations (including structuralised anamnesis and also an element of subjective examination), also an analysis of factors such as: gender, obesity, tobacco smoking, duration of bronchial asthma, allergy to house dust mites, animal fur, mould spores, cockroaches allergens, hypersensitivity to non-steroid anti-inflammatory drugs (NSAIDs), etc., in order to determine their role in the development of resistance to glucocorticoids, as well as to establish whether they are primary or secondary to genetic factors. The detailed information was obtained from medical records of particular patients. The intranasal provocation test was performed with the use of the "spray" method by administering standardized test solutions of 0.04-0.05 ml, through a spray nozzle supplied by the manufacturer and approved for distribution/use. Test solutions were prepared as follows: 1st provocation: dilution 1:10,000 (or more in highly sensitive patients), 2nd provocation: dilution 1:1000, 3rd provocation: dilution 1:100, 4th provocation: dilution 1:10 and 5th provocation: undiluted test solution. The spray nasal allergen challenge was performed according to the approved for distribution/use protocol No. 9531, which is available on the manufacturer's website. [24][25][26]

| Methacholine challenge test
The provocation was performed according to the "ERS technical standard on bronchial challenge testing: general considerations and performance of methacholine challenge tests". First, the patient performed basic spirometry, and then the patient inhaled, using a dispenser, a gradually increasing amount of the substance causing bronchospasm-methacholine. Forced Expiratory Volume (FEV1) change from baseline was assessed. A reduction in FEV1 of ≥20% was considered significant and the triggering concentration (provocative concentration [PC20]) or the provocative dose (PD20) was determined for this value.   For the purpose of internal control, the β-2 microglobulin (β-2M) gene was used, which demonstrates expression at a constant level in the tested samples. Appropriate TaqMan probes that do not react with genomic DNA were chosen for the eight genes and the control gene (β-2M), selected for the analysis. They are presented in Table 1. The experiment involved carrying out a preliminary optimization of the reaction conditions followed by checking the expression of the studied genes in all samples. A detailed description of the RT-qPCR reaction conditions is presented in Table 2.
Assays were performed in two repetitions for each sample.
Averaged Ct values for the two replicates make up the results of the study. Ct is the number of amplification cycles of the PCR product in which the fluorescence level of the dye exceeds the threshold called the limit cycle. Considering the Ct value, it is possible to analyze the amount of baseline cDNA for the selected genes in the studied sample, and thereby to analyze their expression. 12,13,21,28-31

| Data analysis
Analysis of missing data was included in the investigation. Blood sampling in at least two (out of three) time points was sufficient for a patient to be included in the study.

| RESULTS
120 asthmatic patients were invited to participate in the study. The  Table 4.
We found that both the intranasal allergen challenge (specific agents) and methacholine challenge (non-specific agent) contributed  Table 5.
A data analysis was also performed in order to determine whether the provocation type affected the gene expression over time. However, we did not analyze the influence of specific provocation on the change in gene expression, as shown in Table 6.    Note: Sensitivity analysis 3 was added to account for between-group baseline data difference in age and number of allergens.
Abbreviation: MPK, mitogen-activated protein kinase.   effect of particular provocation is inconclusive. We did not study them separately because there were too few patients in the database to conduct such an analysis.
Interestingly, we noticed an increase in the relative gene expression for MAPK kinase isoforms 1 and 3, after both types of provocation over time. This is an important observation because TGF-β1, through the TGFβRI/TGFβRII receptor (ALK5) and MAPK1/3 kinases, independent of Smad signaling proteins, inhibits collagenase and matrix metalloproteinases gene expression, inhibits MHC type II antigen expression and surfactant synthesis by type II pneumocytes. This changing in the expression of these molecules may significantly reduce the progress of bronchial remodeling. 5,11,12,14,20,21 Analyses of the Smad1 gene expression did not show its significant change after performing an allergen and a methacholine challenge test and exposing the asthmatic patient to either a specific or non-specific factor. However, the results were close to the borderline of statistical significance. Consequently, the results of Smad1 mRNA expression should be considered inconclusive. Therefore, both possibilities should be considered: the change in Smad1 protein expression may be dependent or independent of the ongoing underlying inflammation factors in asthma. If Smad1 expression is related to provocation, this can probably be explained in many ways.
In our opinion, it may be due to the fact that Smad1 receives its signal from a different type of ALK receptor than Smad signal proteins 3, 6, and 7. Indeed, ALK1, 2, 3, and 6 receptors are the main signal transducers from TGF-β1 to Smad1, and not from ALK5. The Smad1 signaling protein may have a different function in response to specific and non-specific provocations than the other studied Smad proteins. 5,10,[17][18][19] We conclude this because of high correlation of Smad1 with other tested genes. However, borderline significance allows for speculative discussion only.
Interesting is the change in the Smad3 mRNA expression after the provocation triggered by irritants in allergen and methacholine provocations. A significant change in the relative expression of the Smad3 gene is known to correlate with activation of the Smad2/3 complex and stimulates the intranuclear Smad2/3/4 protein systems and TF, leading to the activation of target gene transcription, including those responsible for bronchial remodeling in asthma, particularly those of MMPs, PAI-1, CTGF, MCP-1, IL-6, TGF-β, TSP-1, TGFR-1/2, fibronectin, proteoglycans, as well as type I and III collagen. 5,[33][34][35] In the above context, the role of Smad6 and Smad7 proteins, which belong to the group of inhibitory proteins (I-Smad) for the F I G U R E 1 mRNA gene expression following provocation over time. Gene expression estimates are expressed as mean −ΔCt values (decrease in the value by a unit means a 2-fold decrease in gene expression) with the whiskers indicating 95% confidence intervals. MET, methacholine provocation; NAS, intranasal provocation F I G U R E 2 Interrelation of mRNA gene expressions in twodimensional representation. Factor 1 retains 51.5% of variance, whereas Factor 2-17.6%. Kaiser-Meyer-Olkin measure = 0.768, the Bartlett's test of sphericity: χ 2 (28) = 960.0, p < 0.0001. The analysis was performed in a fully imputed dataset TGF-β -Smad signaling pathway, is interesting and they respond to signals transmitted by ALK1 and ALK5 receptors. Their role in asthma has not been fully understood. 5,10,18,[36][37][38] In this study, we also performed an exploratory factor analysis of the relative expression change of the studied mRNA genes of the TGF-β-Smad signaling pathway after exposure to provocative factors, as shown in Figure 2. Substantial interrelation of the expressions is a very interesting observation. It shows that there are two factors that determine the cluster change of expression of groups of studied genes of the TGF-β (TGF-β1 and TGF-β3)-Smad (MPK1/3, Smad1/3/6/7) signaling pathway in asthmatic patients. The first leading factor correlates with the majority of genes (except for Smad7 and TGF-β1). The other one is independent of most of these genes, however, it relates to Smad7 and TGF-β1 in an opposing way.
This interrelation also can be observed by analyzing similar trend lines for the studied gene expression changes as illustrated in To make the analysis more reliable, we wanted to add that sensitivity analyses to support the robustness of the obtained results.
The observations were described using statistical methods with all appropriate statistical corrections. This fact was included in the Materials and Methods section.

| LIMITATIONS
Observations were made on 75 participants who underwent an intranasal allergen and bronchial methacholine challenge for diagnostic clinical indications, and who, were different in terms of age and the number of allergens (see Table 4). It is a naturalistic observational study, and patients were allocated to groups based on diagnostic and therapeutic indications, but not randomly. Blood was collected from the patients only at three time points: 0 h, 1 h, and 24 h. The patients' blood was used to perform the tests, not any of their tissue material.
Hence, there are no different biological materials that could potentially show differences in the expression of the studied genes that could be used for molecular comparative studies.
Besides, the patients were not prevented from the impact of external environmental factors for a sufficiently long time before specific and non-specific tests were performed. The modifying effect on the results of the experiment could possibly have had many elements. They included potential confounding factors, such as medications taken by patients, disease duration, comorbidities, duration of the challenge itself, analysis carried out at only two time points (0 h, 1 h, and 24 h), smoking, mutations and polymorphic forms tested by genes, the level of oxidative stress and free radicals, and many other environmental factors. Here, we wanted to show some trends and relationships rather than describe the strictly isolated biochemical and molecular reactions.
The role of BMP proteins, which can modify the expression of Smad1/5 and Smad4 by interacting with ALK1,2,3,6 receptors and interfering with the mRNA expression of the studied genes, was not taken into account either, which had been assumed to be included in this research project. Multicentre experiments on comparable in vitro, animal, and in vivo models, including several blood collections in patients at different time intervals after activation of standardized doses of specific and non-specific irritants would be a valuable addition to our work.
Apart from the limitations presented in the manuscript by the authors above, gene expression was examined without reference to their protein products and it is not always the level of gene expression corresponds to/correlates with the amount of protein product.
Further research is needed at the protein level.

| CONCLUSION
Our experiment allows us to conclude that both examined types of challenges contributed to changes in the relative expression of majority of genes of the TGF-β (TGF-β1 and TGF-β3)-Smad (MPK1/3, Smad1/3/6/7) signaling pathway in asthmatic patients. A significant change in the mRNA expression of the TGF-β1-MPK1/3, and Smad3/ 6/7 genes occurred after an intranasal allergen and bronchial methacholine challenge. TGF-β1 expression increased after methacholine and allergen provocation, which is the main factor activating the entire signaling pathway in asthma, which, we believe, may be clinically useful. Activation of ALK1 and 5 receptors by TGF-β1 is followed by stimulation of Smad-independent and MAPK (Smadindependent) proteins, which is a leading factor responsible for bronchial remodeling in asthma. The discovered change in the mRNA I-Smad expression in asthma still remains unknown and requires further scientific studies. The cluster change in the expression of groups of TGF-β-Smad signaling pathway genes, studied in asthmatic patients (cluster 1: TGF-β1 and Smad7, and cluster 2: TGF-β3, MPK1/3, Smad1/3/6), may be a reason for clustering genetic elements into asthma phenotypes and conducting a deeper analysis of the similarity of interactions between different groups of proteins PANEK ET AL.